Current medical technology allows the use of several different types of tissue for transplantation to correct congenital, diseased-induced or degenerative failure of a recipient's tissue. Some examples include allograft human heart valves, veins, corneas, bone marrow, etc. Investigators have generally agreed that fresh tissue gives improved performance over old or dead tissue.
Human tissue remains viable in vitro for short periods of time, e.g., usually less than two to three days. Storage periods of this limited duration are usually inadequate for most tissue types due to the complexities in assuring the best match of donor to recipient (e.g. such factors as relative size of a graft, human leukocyte antigen and ABO blood group), as well as the time needed to test the tissue for pathogens. Consequently, much of the available donor tissue is unused due to the severe loss of cell viability over time. These shortcomings may be circumvented by the viable cryopreservation and ultracold storage of the tissue.
Ultracold storage of cells and tissues became possible after the discovery in 1949, by Polge, Smith and Parks, that glycerol could be used to protect cells from injury due to freezing. With the advent of low temperature biology, workers in medical and biological fields have been seeking better ways to maintain the viability of frozen donor cells or tissues.
Several methods for freezing cells and cell aggregates have been reported. For example, U.S. Pat. No. 3,303,662 discloses a process for cell preservation that utilizes a cryoprotectant in the freezing process.
The performance of a cryopreserved, transplantable tissue correlates directly with the viability of that tissue upon thawing. One parameter that provides an assessment of the cellular viability of tissue is the general metabolic energy status of the cells. In order for transplanted cells to perform their critical roles in the recipient, these cells must have sufficient metabolic capacity to carry out key energy-dependent processes. For example, one such process that is dependent on cellular metabolic energy is the biosynthesis of proteins. Furthermore, essentially any cellular, tissue or organ function is ultimately dependent on energy derived from cellular metabolism.
Cells that are metabolically and functionally suppressed after thawing may not recover sufficiently to endure the shock of transplantation into a donor, and thus may not survive.
There are several steps in the handling of human tissue for cryopreservation that can decrease the metabolic energy status and depress the energy-dependent functions of the cells. The time between death and the harvest of the tissue (warm ischemia) and the time from harvest until cryopreservation (cold ischemia) are most influential. Prolonged warm and/or cold ischemia results in cells that are severely metabolically and functionally depressed.
Cryopreservation itself appears to reduce cellular energy and metabolic capacity, and to reduce energy-dependent functions at least minimally. Hence, there is a long-standing need for a method of maintaining tissue viability post-implant and for revitalizing cells in the tissue post-harvest, such that the cells essentially completely recover from the transient metabolic lesions and loss of function induced by warm and cold ischemia. The invention therefore fulfills this long-term need for greatly improved viability, and maximizes the functional capacity of cryopreserved cells upon thawing and transplantation. In addition, revitalized cells are better able to withstand the rigors of cryopreservation.
Tissues are currently placed into solutions such as tissue culture media, Lactated Ringers, saline or Collins solution on wet ice for shipping. The concentration of compounds contained in these solutions, the time period during which the tissues are retained therein, and the temperature at which the tissues are shipped can vary widely. Due to the combined effects of these variables, and due to variations in the times of warm and cold ischemia, it is difficult to predict the degree of metabolic and functional depression for any given tissue. One important feature of the present invention is that the method improves the metabolic status, and hence the capacity to function of tissues upon transplant, even with widely varying degrees of metabolic and functional suppression.
Accordingly, it is one object of the present invention to provide a method for revitalizing cells or tissues prior to cryopreservation.
It is another object of the present invention to provide a method of enhancing transplant cell viability and functional capacity upon thawing.
It is yet another object of the present invention to provide a method that improves the ability of a cryopreserved tissue or cell to survive and function upon thawing and transplantation.
It is yet another object of the present invention to provide a method for cell revitalization that can be used concomitantly with other procedures, such as antibiotic sterilization, which may be necessary in the preparation of transplantable tissue for cryopreservation.
These and other objects, features, and advantages of the present invention will become apparent after review of the following detailed description of the disclosed embodiments and the appended claims.